Devalaraja M N, Wang D Z, Ballard D W, Richmond A
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37212-2637, USA.
Cancer Res. 1999 Mar 15;59(6):1372-7.
The basal transcription of the CXC chemokine, melanocyte growth stimulatory activity (MGSA)/growth-regulated protein (GRO)-alpha, is up-regulated in Hs294T melanoma cells compared with the normal retinal pigment epithelial (RPE) cells. Previous studies characterized a cytokine-inducible, functional nuclear factor (NF)-kappaB consensus element in the immediate 5' regulatory region of the MGSA/GRO-alpha gene at -78 bp. Although the cytokine-inducible mechanisms for transcription of this gene are fairly well delineated, the mechanisms involved in its basal up-regulation of transcription in Hs294T melanoma cells are poorly understood. Recently, we demonstrated an increased rate of IkappaB-alpha degradation in Hs294T cells, which leads to an increased nuclear localization of NF-kappaB (R. L. Shattuck-Brandt and A. Richmond. Cancer Res., 57: 3032-3039, 1997). Here we demonstrate that Hs294T melanoma cells have elevated basal IkappaB kinase (IKK) activity relative to RPE cells, causing an increased constitutive IkappaB-alpha phosphorylation and degradation. We also show here that the resultant elevated nuclear NF-kappaB (p50/p65) in these cells is responsible for the increased basal transcription of MGSA/GRO-alpha. Pretreatment of Hs294T or RPE cells with proteasome inhibitors MG115 or MG132 captures the slower migrating, constitutively phosphorylated form of IkappaB-alpha in Hs294T melanoma cells, but not in RPE cells. In addition, a phospho-specific antibody that specifically recognizes the inhibitory form of IkappaB that is phosphorylated at Ser-32 reacted with IkappaB-alpha in Hs294T cell, but not in unstimulated RPE cells. Although the basal level of protein expression of IKK-alpha or IKK-beta are the same in both Hs294T and RPE cells, immunoprecipitation with IKK-alpha antibody combined with activity assay reveal a constitutively active IKK complex in Hs294T melanoma cells. Cotransfection of a 350-bp MGSA/GRO-alpha promoter-luciferase reporter construct with either the dominant negative IKK-alpha or the repressors of NF-kappaB, the IkappaB-alpha wild type or mutants lacking the inducible phosphorylation sites, demonstrates that the increased basal MGSA/GRO-alpha transcription in the Hs294T cells is due to the enhanced nuclear activation of NF-kappaB.
与正常视网膜色素上皮（RPE）细胞相比，Hs294T黑色素瘤细胞中CXC趋化因子黑素细胞生长刺激活性（MGSA）/生长调节蛋白（GRO）-α的基础转录上调。先前的研究在MGSA/GRO-α基因紧邻5'端-78bp的调控区域鉴定出一个细胞因子诱导型功能性核因子（NF）-κB共有元件。尽管该基因转录的细胞因子诱导机制已相当明确，但对其在Hs294T黑色素瘤细胞中基础转录上调所涉及的机制了解甚少。最近，我们证明了Hs294T细胞中IkappaB-α降解速率增加，这导致NF-κB核定位增加（R.L.Shattuck-Brandt和A.Richmond。癌症研究，57:3032 - 3039,1997）。在此我们证明，相对于RPE细胞，Hs294T黑色素瘤细胞具有升高的基础IkappaB激酶（IKK）活性，导致组成型IkappaB-α磷酸化和降解增加。我们还在此表明，这些细胞中由此产生的升高的核NF-κB（p50/p65）负责MGSA/GRO-α基础转录的增加。用蛋白酶体抑制剂MG115或MG132预处理Hs294T或RPE细胞，可捕获Hs294T黑色素瘤细胞中迁移较慢的、组成型磷酸化形式的IkappaB-α，但在RPE细胞中则不能。此外，一种磷酸特异性抗体，可特异性识别在Ser-32处磷酸化的抑制性形式的IkappaB，在Hs294T细胞中与IkappaB-α发生反应，但在未刺激的RPE细胞中则不反应。尽管IKK-α或IKK-β的蛋白质表达基础水平在Hs294T和RPE细胞中相同，但用IKK-α抗体进行免疫沉淀并结合活性测定，揭示了Hs294T黑色素瘤细胞中组成型活性IKK复合物。用350bp的MGSA/GRO-α启动子-荧光素酶报告构建体与显性负性IKK-α或NF-κB抑制剂、IkappaB-α野生型或缺乏诱导性磷酸化位点的突变体共转染，表明Hs294T细胞中基础MGSA/GRO-α转录增加是由于NF-κB核激活增强所致。
